Recent evidence suggests that telomere length maintenance is a crucial determinant of the proliferative potential of mouse and human cells. We will use a genetic approach to dissect the function of the mammalian telomerase reverse transcriptase, TERT, the telomerase-assocated protein, TEP1, and other novel telomerase-associated proteins in vivo. Gene targetting of both TEP1 and TERT in mice will be used to generate embryonic cell lines and adults that completely lack TEP1 and/or TERT. An analysis of telomerase activity, telomere length, and cell proliferation will be carried out in cell lines and mice lacking TEP1 and TERT. Mice deficient in TEP1 and TERT will be crossed into other genetic backgrounds, such as mice with shorter telomeres (mus spretus) and mice with proliferative disorders (such as p16-/-, BRCA2 -/-, p53 -/-) to accentuate possible phenotypes in telomere maintenance and/or proliferative potential. In analogous studies, we will target the disruption of TEP1 and other novel telomerase-associated proteins in normal human diploid fibroblasts that have been reconstituted with telomerase activity. Finally, we will determine whether the subcellular localization of the telomerase components is regulated during cell proliferation and senescence. These approaches will enable us to determine the role of catalytic and non-catalytic telomerase associated proteins in telomere length maintenance in mouse and human cells. This research is an essential step towards the manipulation of telomere length and lifespan of normal and diseased human cells in vivo.